Genetic engineering techniques to introduce and integrate exogenous nucleic acids into a host cell genome are needed in a variety of fields. For example, in the field of synthetic biology, the fabrication of a genetically modified strain requires the insertion of customized DNA sequences into a chromosome of the host cell, and commonly, industrial scale production requires the introduction of dozens of genes into the host organism. Optimized designs for the industrial strain are arrived at empirically, requiring construction and in vivo testing of many DNA assemblies, alone and/or in concert with other biosynthetic pathway components.
Genetic engineering is highly reliant on gene targeting, which utilizes an extrachromosomal fragment of donor template DNA and invokes a cell's homologous recombination (HR) machinery to exchange a chromosomal sequence with an exogenous donor sequence. See, e.g., Capecchi, Science 244:1288-1292 (1989). Gene targeting is limited in its efficiency; in plant and mammalian cells, only ˜1 in 106 cells provided with excess template sequences undergo the desired gene modification. Yeast demonstrates an increased capacity for homologous recombination. However, the successful incorporation of exogenous DNA into yeast genomes is still a comparatively rare event (˜1 in 105), and requires the use of a selectable marker to screen for recombinant cells which usually comprise only a single genomic modification. In addition, since only a limited cache of selectable markers are available for use in yeast, selectable marker(s) must be removed from a recombinant strain to allow for additional genomic modifications using the same markers, and in some instances, prior to releasing the host cell in a manufacturing or natural environment. Thus, independent of the efficiency at which integration can be achieved at any single locus, the one-at-a-time serial nature of genomic engineering requires that making changes at multiple loci requires as many engineering cycles as there are loci to be modified.
The efficiency of gene targeting can be improved when combined with a targeted genomic double-stranded break (DSB) introduced near the intended site of integration. See e.g., Jasin, M., Trends Genet 12(6):224-228 (1996); and Urnov et al., Nature 435(7042):646-651 (2005). So called “designer nucleases” are enzymes that can be tailored to bind to a specific “target” sequence of DNA in vivo and introduce a double-strand break thereto. Such targeted double-strand breaks can be effected, for instance, by transforming a host cell with a plasmid containing a gene that encodes the designer nuclease. The host cell repairs these double-strand breaks by either homology-directed DNA repair or non-homologous end joining. In the course of the repair, either mechanism may be utilized to incorporate an exogenous donor DNA at the target site. If the nuclease is introduced into the cell at the same time as the donor DNA is introduced, the cell can integrate the donor DNA at the target loci.
The advent of designer nucleases has enabled the introduction of transgenes into particular target loci in crops (Wright et al., Plant J 44:693-705 (2005)), to improve mammalian cell culture lines expressing therapeutic antibodies (Malphettes et al., Biotechnol Bioeng 106(5):774-783 (2010)), and even to edit the human genome to evoke resistance to HIV (Urnov et al., Nat Rev Genet 11(9):636-646 (2010)). While impactful, DSB-mediated HR has yet to be exploited to reduce the multiple rounds of engineering needed to integrate multiple DNA assemblies, for example, towards the construction of functional metabolic pathways in industrial microbes.
Thus, there exists a need for methods and compositions that allow for the simultaneous integration of a plurality of exogenous nucleic acids into specific regions of a host cell genome.